Defining the regulatory mechanisms that generate the wide array of neuronal phenotypes in the central nervous system is a critical challenge for the field of development and has important implications for the etiology of neurological disease and neurodegeneration. Neuronal phenotypes are defined by the expression of specific genes, but the mechanisms that regulate transcription of these genes are largely undefined. To address this critical gap in our knowledge for olfactory bulb (OB) juxtaglomerular (JG) neurons that have a dual dopamine and GABA neurotransmitter phenotype, this proposal will establish the molecular mechanisms regulating the co-expression of tyrosine hydroxylase (Th) and glutamic acid decarboxylase 1 (Gad1) genes. The TH and GAD1 enzymes are necessary for the biosynthesis of dopamine and GABA neurotransmitters, respectively. This proposal will test the hypothesis that recruitment of partially overlapping combinatorial sets of transcription factors by novel upstream enhancer regions activate Th and Gad1 transcription in the OB. This proposal will use a combined phylogenetic and proteomic methodology that is a novel approach for establishing the regulatory mechanisms for Th and Gad1 expression. Since critical cis- regulatory elements typically show evolutionary conservation, upstream regions of both Th and Gad1 from several mammalian species will be analyzed by phylogenetic methods. In vitro and in vivo methods will demonstrate that conserved genomic elements are necessary to regulate expression in the mouse OB. Mass spectrometry and proteomic methods will identify the transcription factors that bind to the conserved elements, and functional studies will show that these proteins regulate either Th or Gad1 transcription in the mouse OB. Specific Aim 1 will show that transcription factor proteins bound to conserved Th upstream regions synergistically enhance gene expression mediated by the Th proximal promoter. Preliminary studies have identified hnRNP-A1 as a novel regulatory factor recruited by the Th proximal promoter, validating the combined phylogenetic and proteomic methodology for identifying novel regulatory factors and their binding sequences. Specific Aim 2 will identify the transcription factor proteins bound to a novel and conserved Gad1 upstream region, and demonstrate that these proteins are necessary for Gad1 expression in the OB. These findings will show that some proteins necessary for Gad1 expression are also necessary for Th expression in the OB, and thus, provide a mechanistic basis for the co-expression of Th and Gad1 in a subset of OB JG neurons. This proposal will significantly advance the mechanistic understanding of both OB DA neuronal differentiation and the generation of neuronal phenotypic diversity. PUBLIC HEALTH RELEVANCE: This research proposal is designed to elucidate the molecular mechanisms that regulate the diversity of neuron phenotypes in the central nervous system. This fundamental knowledge can provide critical insight into the molecular basis of neurological and neurodegenerative diseases as well as impact the development of novel therapeutic strategies to treat these syndromes.